The present invention relates to the cleaning of substrates, generally reusable substrates, in order to remove adsorbed prion infectivity. It particularly involves the cleaning of reusable chromatography columns employed during the processes involved in the fractionation of blood plasma. However, it also applies to the cleaning of other substrates, such as surgical instruments etc.
Prion-related disease is believed to be due to prion proteins having altered three-dimensional structure. However, the identification of the infective prion agent has not yet been conclusively resolved. In the present specification we refer to “prion infectivity” as covering the infective agent causative of prion-related disease (whatever that may be). The experimental methods described herein in fact measure prion infectivity in mice.
It is well known that prion infectivity adheres strongly to substrates by a unknown mechanism and means for reliably cleaning such substrates have hitherto been unknown as they will not normally withstand the severe conditions recommended for the inactivation of prion agents such as treatment with 2 M sodium hydroxide combined with heating at 121-138° C. (Taylor D M. Inactivation of prions by physical and chemical means. J Hospital Infection 1999; 43 Suppl.: S69-76).
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle, first described in the UK in 1987. Cases of BSE have since been discovered in over twenty different countries with almost 184 000 cases reported world-wide, 99% of which have occurred in the UK. The emergence of a new neurodegenerative disorder in humans, named variant Creutzfeldt-Jakob disease (vCJD), was subsequently shown to have been due to the BSE agent having been transmitted to humans, probably via the ingestion of diseased animal tissue. By June 2002, the numbers of confirmed or probable cases of vCJD reported were 122 in the UK, 6 in France and 1 in Italy. Individual cases of vCJD have also been reported in Ireland, Hong Kong and in the USA in persons who were resident in the UK previously. However, in the absence of a suitable diagnostic screening test, the prevalence of asymptomatic vCJD in the UK population or elsewhere is not known.
Although other forms of Creutzeldf-Jakob disease (CJD) have been transmitted iatrogenically by a number of medical procedures there is no evidence of such transmission by blood, blood products or plasma derivatives. Nevertheless, lack of knowledge over the prevalence of asymptomatic vCJD, together with the detection of abnormal prion protein in lympho-reticular tissues of individuals infected with vCJD had led to concern that vCJD may be transmitted by blood products. A risk analysis commissioned by the UK Department of Health concluded that there was a theoretical risk of vCJD being transmitted by plasma derivatives prepared from infected donations.
Considerable work has been done on the study of prions and general background information is provided in Stanley B. Prusiner et al “Some Strategies and Methods for the Study of Prions” Chapter 15, Prion Biology and Diseases, 1999 Cold Spring Harbor Laboratory Press. An assessment of the potential of blood plasma fractionation processes to remove the causative agents of transmissible spongiform encephalopathy (TSE) such as CJD or vCJD is given in P. R Foster, Transfusion Medicine, 1999, 9, 3-14 and a review of prions and blood products is given in P R Foster, Annals of Medicine 2000; 32: 501-513. Work presented in P. R. Foster et al, Vox Sang 2000; 78, 86-95, identified depth filtration, particularly using a Seitz KS80 depth filter as being particularly effective in removing prion proteins from blood plasma fractionations streams. Other attempts to remove prion proteins are described in patent application PCT/FR97/00465 which employs a combination of electrostatic adsorption and chromatography; and patent U.S. Pat. No. 5,808,011 which removes prion infectivity from a solution by adsorption of the prion to a chromatography column by use of a pH gradient.
Thus, whilst there is a growing understanding of ways in which prions can be removed from process streams, particularly those involved in blood fractionation, the fate of prion infectivity remains uncertain. Thus, there is a serious risk that prion agents immobilised on chromatography columns during the processing of one batch of blood plasma, may remain during the processing of subsequent batches leading to possible contamination thereof It is therefore important to be able to know with certainty that reusable chromatography columns (which may have a reusable lifetime of several years) can be reliably cleaned of prion infectivity at the end of each batch run. Furthermore, there is a concern that other medical or surgical substrates, such as surgical instruments may also be effective in transmitting CJD. Flechsig E. et al, Mol. Med 2001 October; 7(10): 679-684 reports that prions are readily and tightly bound to stainless steel surfaces and that electrodes used intracerebrally on CJD patients transmitted the disease to two further patients and finally to a chimpanzee, despite attempted disinfection. The UK Department of Health has also assessed the risk of prion infectivity being transmitted to patients via instruments used in surgery and other invasive medical procedures, concluding that “variant and sporadic CJD may be transmitted on surgical instruments” and “current procedures for decontaminating surgical instruments between uses cannot be guaranteed to eliminate the abnormal prion proteins that are thought to be responsible for the transmission of CJD.” (CJD Incidents Panel. Management of possible exposure to CJD through medical procedures: a consultation paper. Department of Health Publications, October 2001). Similarly, there is concern that prion diseases may be transmitted during eye surgery as opthalmic instuments can remain contaminated with debris after cleaning (Dinakaran S, Kayarkar V V. Eye 2002; 16: 281-284). There is therefore a need to reliably clean substrates involved in medical or surgical procedures on patients, so as to avoid the transmission of prion-related disease.